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BMP-2-induced Runx2 Expression Is Mediated by Dlx5, and TGF-β1 Opposes the BMP-2-induced Osteoblast Differentiation by Suppression of Dlx5 Expression

2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês

10.1074/jbc.m211386200

ISSN

1083-351X

Autores

Mi‐Hye Lee, Youn Jeong Kim, Hyun‐Jung Kim, Hyun-Dong Park, A H Kang, Hee‐Moon Kyung, Jae-Hyun Sung, John M. Wozney, Hyun‐Jung Kim, Hyun‐Mo Ryoo,

Tópico(s)

Bone and Dental Protein Studies

Resumo

Intramuscular injection of BMP-2 induces ectopic bone formation in vivo. Similarly, BMP-2 treatment blocks myogenic differentiation and induces osteoblastic transdifferentiation of premyoblastic C2C12 cells. Previous reports suggested that BMP-2-stimulated Runx2 expression could play a pivotal role in transdifferentiation. However, increased Runx2 expression by TGF-β1 did not support osteoblast differentiation in vitro. These results indicate that the induction of Runx2 is not sufficient to explain the BMP-induced transdifferentiation. We found that Dlx5 is specifically expressed in osteogenic cells, and is specifically induced by BMP-2 or -4 signaling but not by other osteotrophic signals or other TGF-β superfamily members. Cycloheximide treatment indicated that Dlx5 was immediately induced by BMP signaling, while Runx2 required de novo protein synthesis. In addition, blocking or overexpressing each transcription factor indicated that Dlx5 is an indispensable mediator of BMP-2-induced Runx2 expression but is not involved in TGF-β1-induced Runx2 expression. Moreover, TGF-β1 opposed BMP-2-induced osteogenic transdifferentiation through Dlx5 suppression by de novo induction of AP-1. Taken together, these results indicate that Dlx5 is an indispensable regulator of BMP-2-induced osteoblast differentiation as well as the counteraction point of the opposing TGF-β1 action. Intramuscular injection of BMP-2 induces ectopic bone formation in vivo. Similarly, BMP-2 treatment blocks myogenic differentiation and induces osteoblastic transdifferentiation of premyoblastic C2C12 cells. Previous reports suggested that BMP-2-stimulated Runx2 expression could play a pivotal role in transdifferentiation. However, increased Runx2 expression by TGF-β1 did not support osteoblast differentiation in vitro. These results indicate that the induction of Runx2 is not sufficient to explain the BMP-induced transdifferentiation. We found that Dlx5 is specifically expressed in osteogenic cells, and is specifically induced by BMP-2 or -4 signaling but not by other osteotrophic signals or other TGF-β superfamily members. Cycloheximide treatment indicated that Dlx5 was immediately induced by BMP signaling, while Runx2 required de novo protein synthesis. In addition, blocking or overexpressing each transcription factor indicated that Dlx5 is an indispensable mediator of BMP-2-induced Runx2 expression but is not involved in TGF-β1-induced Runx2 expression. Moreover, TGF-β1 opposed BMP-2-induced osteogenic transdifferentiation through Dlx5 suppression by de novo induction of AP-1. Taken together, these results indicate that Dlx5 is an indispensable regulator of BMP-2-induced osteoblast differentiation as well as the counteraction point of the opposing TGF-β1 action. Bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic protein; TGF, transforming growth factor; Dlx5-AS, Dlx5 antisense; ALK3 QD, constitutive active BMPR IA; ALK 6 QD, constitutive active BMPR IB; ALK3 KR, dominant negative BMPR IA; ALK6 KR, dominant negative BMPR IB; ALP, alkaline phosphatase; OC, osteocalcin; Col I, type I collagen; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHX, cycloheximide.1The abbreviations used are: BMP, bone morphogenetic protein; TGF, transforming growth factor; Dlx5-AS, Dlx5 antisense; ALK3 QD, constitutive active BMPR IA; ALK 6 QD, constitutive active BMPR IB; ALK3 KR, dominant negative BMPR IA; ALK6 KR, dominant negative BMPR IB; ALP, alkaline phosphatase; OC, osteocalcin; Col I, type I collagen; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHX, cycloheximide. were originally identified from demineralized bone matrix as factors that induce ectopic bone formation when implanted into muscular tissue (1Urist M.R. Science. 1965; 150: 893-899Crossref PubMed Scopus (4449) Google Scholar). In vitro models of bone formation by BMP have also been established using myoblastic lineage cells (2Yamaguchi A. Katagiri T. Ikeda T. Wozney J.M. Rosen V. Wang E.A. Kahn A.J. Suda T. Yoshiki S. J. Cell Biol. 1991; 113: 681-687Crossref PubMed Scopus (653) Google Scholar, 3Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1283) Google Scholar). Using this system, the molecular mechanism of the BMP-2-induced ectopic bone formation has been investigated. BMP-2 is not only a potent inducer of osteogenesis. It can also block the differentiation of C2C12 myoblasts into mature muscle cells by suppressing the master control genes for myoblast differentiation (3Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1283) Google Scholar). Subsequently, expression of osteoblast phenotypic marker genes, such as alkaline phosphatase and osteocalcin, is induced by continuous BMP-2 treatment of C2C12 cells (3Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1283) Google Scholar, 4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar). BMPs exert their diverse biological effects through two types of transmembrane receptors; BMP receptor type I (BMPR-I) and type II (BMPR-II), which possess intrinsic serine/threonine kinase activity (5Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). BMPR-I is further subclassified into BMPR-IA (also called ALK3) and BMPR-IB (also called ALK6). It has been shown that both the inhibition of myoblast differentiation and the induction of osteoblast differentiation by BMP-2 involve the activations of BMPR-I receptors (6Namiki M. Akiyama S. Katagiri T. Suzuki A. Ueno N. Yamaji N. Rosen V. Wozney J.M. Suda T. J. Biol. Chem. 1997; 272: 22046-22052Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Akiyama S. Katagiri T. Namiki M. Yamaji N. Yamamoto N. Miyama K. Shibuya H. Ueno N. Wozney J.M. Suda T. Exp. Cell Res. 1997; 235: 362-369Crossref PubMed Scopus (94) Google Scholar), their intracellular transducers Smad1 and Smad5 (8Yamamoto N. Akiyama S. Katagiri T. Namiki M. Kurokawa T. Suda T. Biochem. Biophys. Res. Commun. 1997; 238: 574-580Crossref PubMed Scopus (200) Google Scholar, 9Nishimura R. Kato Y. Chen D. Harris S.E. Mundy G.R. Yoneda T. J. Biol. Chem. 1998; 273: 1872-1879Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), and the osteogenic master transcription factor Runx2 (4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar, 10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar).Runt-related transcription factor 2 (Runx2), previously known as Cbfa1/Pebp2αA/AML3, plays an essential role in osteoblast differentiation (11Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3605) Google Scholar). Runx2 knockout mice display complete absence of bone due to arrested osteoblast maturation (12Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar). Our previous results demonstrated that Runx2 plays a central role in BMP-2- and TGF-β1-induced transdifferentiation of C2C12 cell at an early restriction point to divert the cells from the myogenic pathway. However, commitment and progression of osteogenesis appeared to require interactions with the BMP-2 signaling machinery, but not with TGF-β1 (4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar). A subsequent study suggested that BMP-specific Smads might play an indirect role in inducing Runx2 and that an additional step of de novo protein synthesis was required (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar). Moreover, it has been suggested that BMP-specific Smads interact with the Runx2 protein and that the interaction between Runx2 and BMP-specific Smads could determine ligand-specific activities (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar, 13Zhang Y.W. Yasui N. Ito K. Huang G. Fujii M. Hanai J. Nogami H. Ochi T. Miyazono K. Ito Y. Proc. Natl. Acad. Sci. 2000; 97: 10549-10554Crossref PubMed Scopus (308) Google Scholar). Although both BMP-2 and TGF-β1 stimulate Runx2 expression, only BMP-2 induces osteogenic marker genes. Therefore, Runx2 alone is insufficient to mediate BMP-induced osteoblast differentiation but requires collaboration with other signaling molecules that are stimulated by BMP signaling. This study addresses two unsolved questions concerning the mechanism of BMP-2-induced osteoblast differentiation. First, what is the protein (possibly a transcription factor) that mediates the stimulation of the Runx2 expression in response to BMP-specific Smads activation (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar)? Second, both TGF-β1 and BMP-2 commonly stimulate Runx2 expression, however, TGF-β1 opposes the stimulated osteogenic differentiation by BMP-2. To resolve these questions, we have explored the possible involvement of Dlx5 in BMP-2-induced osteoblast differentiation because Dlx5 is induced by BMP-2 treatment (14Miyama K. Yamada G. Yamamoto T.S. Takagi C. Miyado K. Sakai M. Ueno N. Shibuya H. Dev. Biol. 1999; 208: 123-133Crossref PubMed Scopus (173) Google Scholar, 15Luo T. Matsuo-Takasaki M. Lim J.H. Sargent T.D. Int. J. Dev. Biol. 2001; 45: 681-684PubMed Google Scholar) and is known to play crucial roles in osteoblast differentiation (14Miyama K. Yamada G. Yamamoto T.S. Takagi C. Miyado K. Sakai M. Ueno N. Shibuya H. Dev. Biol. 1999; 208: 123-133Crossref PubMed Scopus (173) Google Scholar, 16Shirakabe K. Terasawa K. Miyama K. Shibuya H. Nishida E. Genes to Cells. 2001; 6: 851-856Crossref PubMed Scopus (156) Google Scholar, 17Ryoo H.M. Hoffmann H.M. Beumer T. Frenkel B. Towler D.A. Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Mol. Endocrinol. 1997; 11: 1681-1694Crossref PubMed Scopus (217) Google Scholar, 18Tadic T. Dodig M. Erceg I. Marijanovic I. Mina M. Kalajzic Z. Velonis D. Kronenberg M.S. Kosher R.A. Ferrari D. Lichtler A.C. J. Bone Miner. Res. 2002; 17: 1008-1014Crossref PubMed Scopus (69) Google Scholar).Dlx5 is a bone inducing transcription factor that is expressed in later stages of osteoblast differentiation (17Ryoo H.M. Hoffmann H.M. Beumer T. Frenkel B. Towler D.A. Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Mol. Endocrinol. 1997; 11: 1681-1694Crossref PubMed Scopus (217) Google Scholar). Forced expression of Dlx5 leads to osteocalcin expression and a fully mineralized matrix in cell culture (14Miyama K. Yamada G. Yamamoto T.S. Takagi C. Miyado K. Sakai M. Ueno N. Shibuya H. Dev. Biol. 1999; 208: 123-133Crossref PubMed Scopus (173) Google Scholar, 18Tadic T. Dodig M. Erceg I. Marijanovic I. Mina M. Kalajzic Z. Velonis D. Kronenberg M.S. Kosher R.A. Ferrari D. Lichtler A.C. J. Bone Miner. Res. 2002; 17: 1008-1014Crossref PubMed Scopus (69) Google Scholar). Normally, Dlx5 expression is detected in discrete neuronal tissues and developing skeletal elements such as cartilage, bone, and tooth (19Simeone A. Acampora D. Pannese M. D'Esposito M. Stornaiuolo A. Gulisano M. Mallamaci A. Kastury K. Druck T. Huebner K. Boncinelli E. Proc. Natl. Acad. Sci. 1994; 91: 2250-2254Crossref PubMed Scopus (260) Google Scholar, 20Zhao G.Q. Zhao S. Zhou X. Eberspaecher H. Solursh M. de Crombrugghe B. Dev. Biol. 1994; 164: 37-51Crossref PubMed Scopus (77) Google Scholar). Moreover, Dlx5-deficient mice demonstrate severe craniofacial abnormalities with a delayed ossification of the cranium and abnormal osteogenesis (21Acampora D. Merlo G.R. Paleari L. Zerega B. Postiglione M.P. Mantero S. Bober E. Barbieri O. Simeone A. Levi G. Development. 1999; 126: 3795-3809Crossref PubMed Google Scholar), and Dlx5/Dlx6 double knockout mouse showed much more comprehensive bone defects (22Robledo R.F. Rajan L. Li X. Lufkin T. Genes Dev. 2002; 16: 1089-1101Crossref PubMed Scopus (321) Google Scholar). These results strongly suggest that Dlx5 plays important and evolutionally conserved roles in the development of mineralized tissues even if there is a functional compensation by other members of the Dlx family. In this study we demonstrate that Dlx5 is the proximal target of BMP-signaling and, in BMP-2-induced osteoblast differentiation, Dlx5 plays a pivotal role in stimulating downstream osteogenic master transcription factor Runx2 which in turn work sequentially and/or work together to induce the expression of bone marker genes that represent transdifferentiation. Furthermore we also show that Dlx5 is the target of the opposing action of TGF-β on BMP-induced osteoblast transdifferentiation.EXPERIMENTAL PROCEDURESMaterials—Bioactive recombinant human BMP-2, -4, growth, and differentiation factor (GDF)-5, -6, -7 (23Wolfman N.M. Hattersley G. Cox K. Celeste A.J. Nelson R. Yamaji N. Dube J.L. DiBlasio-Smith E. Nove J. Song J.J. Wozney J.M. Rosen V. J. Clin. Invest. 1997; 100: 321-330Crossref PubMed Scopus (467) Google Scholar) were from Genetics Institute Inc. (Cambridge, MA). Recombinant human TGF-β1 and fibroblast growth factor (FGF)-2 were purchased from R&D Systems Inc. (Minne-apolis, MN). Vitamin D3, ascorbic acid, dexamethasone, alkaline phosphatase staining kit, and cycloheximide were purchased from Sigma Chemical Company. Recombinant sonic hedgehog protein was generously provided by Dr. Masahiro Iwamoto (24Enomoto-Iwamoto M. Nakamura T. Aikawa T. Higuchi Y. Yuasa T. Yamaguchi A. Nohno T. Noji S. Matsuya T. Kurisu K. Koyama E. Pacifici M. Iwamoto M. J. Bone Miner. Res. 2000; 15: 1659-1668Crossref PubMed Scopus (79) Google Scholar). Superscript™ first-strand synthesis system for reverse transcription and LipofectAMINE plus were from Invitrogen (Carlsbad, CA). Taq polymerase, dNTP mixture, and G418 were from Promega (Madison, WI). Megaprime DNA labeling system kit was from Amersham Biosciences. Express hybridization solution was from Clontech (Palo Alto, CA), and Zetaprobe membrane was from BioRad (Melville, NY).Cell Culture—Mouse myogenic C2C12 cells, osteoblast-like MC3T3-E1 cells, and rat osteosarcoma cell line ROS17/2.8 were maintained as previously described (4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar, 17Ryoo H.M. Hoffmann H.M. Beumer T. Frenkel B. Towler D.A. Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Mol. Endocrinol. 1997; 11: 1681-1694Crossref PubMed Scopus (217) Google Scholar). ST2 cells (murine bone marrow-derived stromal cells) and C3H10T1/2 cells (stem cell-like fibroblasts) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Mouse chondrogenic ATDC5 cells (Riken, Japan) and adipogenic 3T3L1 cells were maintained as previously described (25Shukunami C. Ishizeki K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (251) Google Scholar, 26Olson A.L. Knight J.B. Pessin J.E. Mol. Cell Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (207) Google Scholar). Cells were inoculated at a density of 1 × 106 cells/100 mm culture dishes. To examine the effects of BMP-2 or TGF-β1 on cell differentiation, the cells were cultured for indicated period with or without treatment of indicated amount of the factors, in the respective medium with 5% fetal bovine serum. All cells were harvested with phosphate-buffered saline by scraping with rubber policemen at 4 °C.RNA Extraction and Reverse Transcription—Total cellular RNA was extracted from the cells and the concentration was measured by spectrophotometer (4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar). RNA integrity was assessed by the ratio of 28 S/18 S ribosomal RNA after electrophoresis in 1% agarose/5.5% formaldehyde gels. Reverse transcription was performed with a Superscript™ first-strand synthesis system for RT-PCR. 1 μg of total cellular RNA as a template, 0.5 μg of oligo(dT)12–18, 200 units of Superscript II reverse transcriptase, 0.1 volume of 10× reverse transcription buffer, 0.5 mm of dNTP mixture (each of dATP, dCTP, dGTP, and dTTP), 10 mm of dithiothreitol were used for first strand cDNA synthesis for 60 min at 42 °C. To eliminate contamination by RNA, the reverse-transcribed cDNA mixture was incubated with 2 units of RNase H for 20 min at 37 °C.Polymerase Chain Reaction (PCR) and Northern/Southern Blot Analysis—Oligonucleotides for the PCR of mouse Dlx5 and mouse GAPDH were synthesized (TakaraKorea, Seoul, Korea). The nucleotide sequences of the sense strand are listed in Table I. Dlx5 and GAPDH were PCR amplified with reverse-transcribed cDNA as template, 0.2 μm each forward and reverse primer set, 0.4 mm dNTP mixture (each dATP, dCTP, dGTP, and dTTP), 0.1 volume of 10× PCR buffer, 1 unit of Taq polymerase were used for the reaction. The cycling parameters for Dlx5 was as follows: 95 °C for 40 s, 54 °C for 1 min, 72 °C for 1 min for 35 cycles, followed by 72 °C for 5 min. In the case of GAPDH for internal control, 95 °C for 40 s, 55 °C for 1 min, 72 °C for 1 min for 25 cycles, followed by 72 °C for 5 min. The PCR-amplified products were loaded 1% agarose gel for electrophoresis and checked by ethidium bromide staining. The PCR products were subcloned into pBluescript KS vector and used as a probe for Northern or Southern blot hybridization after sequencing. Northern blot analysis for Dlx5, Runx2, and osteoblast marker genes was performed as previously described (4Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (239) Google Scholar, 10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar). As Dlx5 expression was very low and obscured by 18 S ribosomal RNA in non-osteoblastic cells, it is difficult to detect by Northern blot analysis. Thus, Dlx5 expression in non-osteoblastic cells was determined by RT-PCR. When the RT-PCR results required a quantitative value, the RT-PCR products (less than 25 cycles) were separated on agarose gel and Southern blot analysis was performed.Table IOligonucleotide primers used in the RT-PCR amplificationNameStrandPrimer sequenceAccession #Sequence locationmDlx5F177Sense5′-GACAGGATCCCTATG*ACAGGAGTGTTTGAC-3′U67840177-196mDlx5R1050Antisense5′-TGGACTCGAGATCTA**ATAAAGCGTCCCGGA-3′U678401031-1050mGAPF587Sense5′-GCCACCCAGAAGACTGTGGATGGC-3′M32599587-610mGAPR1033Antisense5′-CATGTAGGCCATGAGGTCCACCAC-3′M325991010-1033 Open table in a new tab Plasmid Constructs—The mDlx5 RT-PCR product was subcloned into pcDNA3.1 (Invitrogen) and was used for the Dlx5 expression construct, pcDNA3.1-Dlx5. The entire coding region of mouse Dlx5 cDNA was amplified by PCR with the Dlx5 primers in Table I. Translation initiation and stop codons in the Dlx5 primers are underlined. To produce Dlx5 antisense expression construct, pCMV5-Dlx5-AS, Dlx5 PCR product was subcloned into pCMV5 expression vector generating antisense Dlx5.Transient Transfection and Establishment of Stable Cell Line—To establish Dlx5 stable cell line (C2C12-pcDNA3.1-Dlx5) and Dlx5 antisense stable cell line (C2C12-pCMV5-Dlx5-AS), C2C12 cells were transfected with pcDNA3.1-Dlx5 and pCMV5-Dlx5-AS Dlx5. Constitutive active BMPR-IA and -IB stable cells were established by transfecting ALK-3 QD and ALK-6 QD expression vector (7Akiyama S. Katagiri T. Namiki M. Yamaji N. Yamamoto N. Miyama K. Shibuya H. Ueno N. Wozney J.M. Suda T. Exp. Cell Res. 1997; 235: 362-369Crossref PubMed Scopus (94) Google Scholar), and dominant negative BMPR-IA and -IB stable cells were established by transfecting ALK-3 KR and ALK-6 KR expression vectors (27Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar) into C2C12 cells, respectively. BMPR-related expression vectors were generously provided by Drs. Heldin and ten Dijke. c-Jun expression vector was a kind gift from Dr. Stein and dominant negative c-Fos (A-fos) expression vector was provided by Dr. Bae (28Lee K.S. Hong S.H. Bae S.C. Oncogene. 2002; 21: 7156-7163Crossref PubMed Scopus (273) Google Scholar). After overnight stabilization in the maintenance medium, stably transfected cell clones were selected with G418. After 2 weeks culture under the selection medium, cell colonies were subcultured. Among the 50 viable G418-resistant cell clones, more than 70% of cells turned out to express the transfected gene. Dlx5 overexpression and antisense blocking in the respective stable cells were confirmed by Western blot analysis. The establishment of Runx2(–/–) calvarial cells, Smad1, Smad5, and Runx2 stable cells was described in our previous report (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar).Alkaline Phosphatase Staining—Alkaline phosphatase (ALP) activity has been widely accepted as a simple and easiest way to determine the osteogenic differentiation, especially in the BMP-2-induced osteogenic transdifferentiation in C2C12 cells (3Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1283) Google Scholar, 6Namiki M. Akiyama S. Katagiri T. Suzuki A. Ueno N. Yamaji N. Rosen V. Wozney J.M. Suda T. J. Biol. Chem. 1997; 272: 22046-22052Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Akiyama S. Katagiri T. Namiki M. Yamaji N. Yamamoto N. Miyama K. Shibuya H. Ueno N. Wozney J.M. Suda T. Exp. Cell Res. 1997; 235: 362-369Crossref PubMed Scopus (94) Google Scholar). Cells were washed with phosphate-buffered saline twice, fixed with 2% paraformaldehyde, and stained for alkaline phosphatase according to the manufacturer's instructions (Sigma).Western Blot Analysis—Proteins from cell lysates or nuclear extracts were resolved by 13% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidine difluoride membrane (Schleicher & Schuell, Dassel, Germany). All procedures of Western blot analysis were done as previously described (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar). Dlx5 antibody was raised against a 19-amino acid peptide located at the C terminus 268–286 of the deduced polypeptide sequence (GenBank™ accession number AAC52843). Anti-Dlx5 antiserum against the synthetic peptide was generated from rabbit and purified by affinity chromatography using IgG (Fc)-specific resin (TakaraKorea, Seoul, Korea). Anti-c-Jun rabbit polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). After primary antibody reaction and washing, the blot was incubated with the horseradish peroxidase-conjugated goat anti-rabbit antibody (Advanced Biochemicals Inc., Seoul, Korea) for 1 h at room temperature. After washing, the signal was detected by ECL plus (Amersham Biosciences).RESULTSDlx5 Is Specifically Expressed in Osteoblastic Cells and Is a Direct and Specific Target of BMP Signaling—Dlx5 expression was examined in osteoblast lineage cells, MC3T3-E1, ROS 17/2.8, and ST2 cells, and in a chondrogenic cell line, ATDC5 cells. In the absence of BMP-2 treatment, Dlx5 was basally expressed in these osteogenic and chondrogenic cells (Fig. 1A). Although basal Dlx5 expression was very strong in ROS 17/2.8 cells as previously reported (17Ryoo H.M. Hoffmann H.M. Beumer T. Frenkel B. Towler D.A. Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Mol. Endocrinol. 1997; 11: 1681-1694Crossref PubMed Scopus (217) Google Scholar) Dlx5 expression was stimulated by BMP-2 treatment but not by TGF-β1. In ST2 cells, Dlx5 expression was even suppressed by TGF-β1 (Fig. 1A). In contrast to these results from osteo-chondrogenic cells, basal Dlx5 expression was undetectable in non-osteoblastic cells, such as myogenic C2C12 cells, fibroblastic C3H10T1/2 cells (10T1/2), and adipogenic 3T3-L1 cells. BMP-2 treatment induced Dlx5 expression in these cells whereas TGF-β1 did not (Fig. 1B).To determine whether Dlx5 is a necessary and commonly employed component for the osteogenic shift of non-osteoblastic stem cells, we treated C2C12 myogenic cells with several osteotrophic hormones or growth factors (Fig. 2A). Vitamin D3 or dexamethasone did not induce Dlx5 expression in these cells nor did osteogenic growth factors such as FGF-2 and sonic hedgehog, or ascorbic acid. Among the TGF-β superfamily members, neither TGF-β1 nor GDF-5, -6, -7 induced Dlx5 expression in C2C12 cells. Only BMP-2 or BMP-4 treatment induced Dlx5 expression. Coincident with Dlx5 expression, alkaline phosphatase activity was strongly activated by BMP-2 or BMP-4 treatment (Fig. 2A). These results indicated that Dlx5 is a specific target of BMP-signaling.Fig. 2Dlx5 is a specific and direct target of BMP-signaling in C2C12 cells. A, BMP-specific expression of Dlx5 in C2C12 cells. The cells were treated with osteogenic growth factors, hormones or TGF-β superfamily members for additional 48 h after reaching visual confluency. Total cellular RNA was purified from the cells and Dlx5 expression was determined by RT-PCR using GAPDH as an internal control. PCR products were separated in 1% agarose gel and visualized by ethidium bromide staining. ALP activity was analyzed by histochemical staining as indicated under “Experimental Procedures.” BMP-2 (200 ng/ml), BMP-4 (200 ng/ml), TGF-β1 (10 ng/ml), GDF5 (200 ng/ml), GDF6 (200 ng/ml), GDF7 (200 ng/ml), FGF2 (10 ng/ml), ascorbic acid (vit C, 50 μg/ml), vitamin D3 (vit D, 10–8m), dexamethasone (Dexa, 10–8m) and Sonic hedgehog (Shh, 300 ng/ml). B, dose-dependent induction of Dlx5 expression by BMP-2 in C2C12. The cells were treated with 3, 30, 90, and 300 ng/ml of BMP-2 for additional 6 h after reaching visual confluency. Dlx5 expression was determined by RT-PCR and subsequent Southern blot analysis. C, time-dependent induction of Dlx5 expression by BMP-2 in C2C12 cells. The cells were treated with 300 ng/ml of BMP-2 for the indicated period after reaching visual confluency. Dlx5 expression was determined as described in B. D, early confluent C2C12 cells were pretreated with 10 μg/ml CHX for 30 min, and then 300 ng/ml of BMP-2 was added for additional 1 h, 3 h, and 1 day. Dlx5 expression was determined as described in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)BMP-2-induced Dlx5 expression was not detected at 3 ng/ml but was dose-dependently induced by 30–300 ng/ml of BMP-2 treatment (Fig. 2B) similar to the levels required to induce alkaline phosphatase (10Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (753) Google Scholar). Dlx5 expression was initially detected at 1 h after BMP-2 treatment, gradually increased, and then continuously detected through 7 days of BMP-2 treatment (Fig. 2C). This time-dependent expression pattern indicates that Dlx5 is an early response gene and could be a direct target of BMP-signaling. In order to confirm the idea, cycloheximide (CHX), a protein synthesis inhibitor, was pretreated for 30 min and then BMP-2 was treated for additional 1 h, 3 h and 1day. The CHX pretreatment could not block BMP-2-induced Dlx5 expression (Fig. 2D), indicating that Dlx5 is the direct target of BMP-signaling.Activation of the BMP-Signaling Components Induced Dlx5 Expression without Treatment of BMP—Activation of BMP signaling is initiated by the binding of the ligand to the BMP receptor II and BMP receptor IA or IB. The hetero-tetramerization of two type I- and two type II-receptors activates the serine/threonine kinase in the cytoplasmic domain of BMPR-I. Next, Smad1 and Smad5 are activated by the receptor kinase through the phosphorylation of their C-terminal serines of SSXS. To determine whether activation of the BMP-signaling components was sufficient to induce Dlx5 expressio

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